Fetal Pulmonary Artery Endothelial Cells Research Articles

Endothelin-1 decreases endothelial NOS expression and activity through ETA receptor-mediated generation of hydrogen peroxide

Similar to infants born with persistent pulmonary hypertension of the newborn (PPHN), there is an increase in circulating endothelin-1 (ET-1) and decreased endothelial nitric oxide synthase (eNOS) gene expression in an ovine model of PPHN. These abnormalities lead to vasoconstriction and vascular remodeling. Our previous studies have demonstrated that reactive oxygen species (ROS) levels are elevated in the pulmonary arteries from PPHN lambs and that ET-1 increases ROS production in pulmonary arterial smooth muscle cells (PASMC) in culture. Thus the objective of this study was to determine whether there was a feedback mechanism between the ET-1-mediated increase in ROS in fetal PASMC (FPASMC) and a decrease in eNOS gene expression in fetal pulmonary arterial endothelial cells (FPAEC). Our results indicate that ET-1 increased H2O2 levels in FPASMC in an endothelin A receptor-dependent fashion. This was observed in both FPASMC monoculture and in cocultures of FPASMC and FPAEC. Conversely, ET-1 decreased H2O2 levels in FPAEC monoculture in an endothelin B receptor-dependent fashion. Furthermore, ET-1 decreased eNOS promoter activity by 40% in FPAEC in coculture with FPASMC. Promoter activity was restored in the presence of catalase. In FPAEC in monoculture treated with 0-100 microM H2O2, 12 microM had no effect on eNOS promoter activity, but it increased eNOS protein levels by 50%. However, at 100 microM, H2O2 decreased eNOS promoter activity and protein levels in FPAEC by 79 and 40%, respectively. These data suggest a role for smooth muscle cell-derived H2O2 in ET-1-mediated downregulation of eNOS expression in children born with PPHN.

Developmental differences in the shear stress-induced expression of endothelial NO synthase: changing role of AP-1.

Endothelial nitric oxide synthase (eNOS) mRNA and protein levels increase during late gestation and then decrease postnatally in sheep lung parenchyma. The increase in fluid shear stress at birth, resulting from increased pulmonary blood flow, is an important mediator of postnatal eNOS gene expression. Our objective was to identify factors stimulating eNOS expression in pulmonary arterial endothelial cells (PAEC) in response to shear stress and to determine if these factors are developmentally regulated. PAEC were isolated from fetal lambs and adult sheep. Transcriptional activity from a 1,600-bp eNOS promoter fragment increased in both fetal and adult PAEC exposed to 8 h of shear stress. Conversely, activity driven from an 840-bp promoter fragment containing a putative activator protein (AP)-1 binding site was increased only in fetal PAEC. This increase was completely abolished in an identical construct containing a mutant AP-1 sequence. The AP-1 protein c-Jun was localized to the cytosol in static adult PAEC and to the nucleus in static fetal PAEC. After shear, c-Jun was nuclear localized in both cell types. However, transcriptionally active phosphorylated c-Jun was elevated only in the nuclei of sheared fetal PAEC. Resting levels of eNOS and NO were 2- and 20-fold higher, respectively, in fetal cells. Shear increased eNOS and NO in both cell types: levels were approximately 2.5-fold higher in fetal PAEC. Phosphorylation of Akt and eNOS was evident in sheared fetal but not adult PAEC. We have therefore identified mechanisms of eNOS regulation at the transcriptional level and to be enzyme activation specific to the fetal pulmonary arterial circulation.

Estrogen acutely activates prostacyclin synthesis in ovine fetal pulmonary artery endothelium.

Prostacyclin (PGI(2)) is a key mediator of pulmonary vasodilation during perinatal cardiopulmonary transition, at a time when fetal plasma estrogen levels are rising. We have previously shown that estradiol-17beta (E(2)) rapidly stimulates nitric oxide production by ovine fetal pulmonary artery endothelial cells (PAEC), and that this occurs through nongenomic mechanisms which are calcium- and tyrosine kinase-mitogen-activated protein (MAP) kinase-dependent. In the present study, we determined if E(2) acutely activates PGI(2) production in PAEC. E(2) (10(-8) M for 15 min) caused a 52% increase in PGI(2), the threshold concentration was 10(-10) M E(2), the effect occurred within 5 min, and it was not related to changes in cyclooxygenase type 1 (COX-1) or COX-2 abundance. Estrogen receptor (ER) alpha and ER beta proteins and mRNAs were found to be constitutively expressed in PAEC, and PGI(2) stimulation with E(2) was fully blocked by both ER antagonism with ICI 182,780, which is not selective for either ER isoform, and the ER beta-specific antagonist RR-tetrahydrochrysene. The rapid response to E(2) was also inhibited by calcium chelation, whereas genistein- or PD98059-induced inhibition of tyrosine kinase and MAP kinase kinase, respectively, had no effect. Thus, E(2) causes rapid stimulation of PGI(2) synthesis in fetal PAEC, this process is mediated by ER beta, and it is calcium-dependent and tyrosine kinase-MAP kinase-independent. These mechanisms may play a role in pulmonary vasodilation in the perinatal period.

Maturational Changes in Ovine Pulmonary Metabolism of Platelet-Activating Factor: Implications for Postnatal Adaptation

We recently reported that PAF acetylhydrolase (PAF-Ah) mRNA level and PAF-Ah activity in lamb lungs are up-regulated in the immediate newborn period, thereby facilitating the fall in postnatal PAF levels as well as a fall in pulmonary vascular resistance (B. O. Ibe, F. C. Sardar, and J. U. Raj, Mol Genet Metab 69:46–55, 2000). We have studied hypoxia effects on PAF synthesis and PAF-Ah activity in fetal lamb pulmonary arterial smooth muscle cells (FPASMC) and endothelial cells (FPAEC). We also studied PAF synthesis by platelets, and PAF-Ah activity in plasma of perinatal lambs at different ages. PAF synthesis (means ± SEM, pmol/106 cells) by SMC in baseline was 168 ± 27 and increased 3-fold on stimulation with A23187. Hypoxia augmented A23187-stimulated PAF synthesis by 30%. In FPAEC, baseline synthesis was 0.54 ± 0.062 and increased 3-fold to 1.72 ± .34. Hypoxia had no effect on PAF synthesis by EC. FPASMC produced over 300-fold more PAF than FPAEC. PAF synthesis by platelets was 47.02 ± 7.1, 63.4 ± 6.6, 71.5 ± 9.9, and 62.2 ± 5.2 for fetal, and newborn lambs <2 h, <1 day, and 6–12 days, old, respectively. PAF synthesis by platelets of <1 day-old lambs was different from that of fetal lambs. PAF-Ah activity (nmol lyso-PAF/min/mg protein) by FPASMC in normoxia was 3.41 ± 0.38 which was 50% higher than the rate in hypoxia. Activity in FPAEC was 1.75 ± 0.37 which was not different from hypoxia. PAF-Ah activity in fetal lamb plasma was 47.83 ± 6.87 which was different from 155.32 ± 12.10, the activity in plasma of newborn <1 day old. Activity in the other perinatal lambs did not differ from fetal or newborn <1 d. Our data suggest that lower pulmonary vascular PAF synthesis in normoxia together with higher PAF-Ah activity during immediate postnatal period is necessary to ensure rapid catabolism of PAF in vivo so as to facilitate postnatal adaptation of the pulmonary and systemic circulations.

Shear stress regulation of endothelial NOS in fetal pulmonary arterial endothelial cells involves PKC.

We have shown that increased pulmonary blood flow at birth increases the activity and expression of endothelial nitric oxide (NO) synthase (eNOS). However, the signal transduction pathway regulating this process is unclear. Because protein kinase C (PKC) has been shown to be activated in response to shear stress, we undertook a study to examine its role in mediating shear stress effects on eNOS. Initial experiments demonstrated that PKC activity increased in response to shear stress. NO production in response to shear stress was found to be biphasic, with an increase in NO release up to 1 h, a plateau phase until 4 h, and another increase between 4 and 8 h. PKC inhibition reduced the initial rise in NO release by 50% and the second increase by 70%. eNOS mRNA and protein levels were also increased in response to shear stress, whereas PKC inhibition prevented this increase. The stimulation of PKC activity with phorbol ester increased eNOS gene expression without increasing NO release. These results suggest that PKC may play different roles in shear stress-mediated release of NO and increased eNOS gene expression.

Cell growth modulates nitric oxide synthase expression in fetal pulmonary artery endothelial cells.

Nitric oxide (NO), produced by endothelial (e) nitric oxide synthase (NOS), is a critical mediator of vascular function and growth in the developing lung. Pulmonary eNOS expression is diminished in conditions associated with altered pulmonary vascular development, suggesting that eNOS may be modulated by changes in pulmonary artery endothelial cell (PAEC) growth. We determined the effects of cell growth on eNOS expression in cultured ovine fetal PAEC studied at varying levels of confluence. NOS enzymatic activity was sixfold greater in quiescent PAEC at 100% confluence compared with more rapidly replicating cells at 50% confluence. To determine if there is a reciprocal effect of NO on PAEC growth, studies of NOS inhibition or the provision of exogenous NO from spermine NONOate were performed. Neither intervention had a discernable effect on PAEC growth. The influence of cell growth on NOS activity was unique to pulmonary endothelium, because varying confluence did not alter NOS activity in fetal systemic endothelial cells. The effects of cell growth induced by serum stimulation were also evaluated, and NOS enzymatic activity was threefold greater in quiescent, serum-deprived cells compared with that in serum-stimulated cells. The increase in NOS activity observed at full confluence was accompanied by parallel increases in eNOS protein and mRNA expression. These findings indicate that eNOS gene expression in fetal PAEC is upregulated during cell quiescence and downregulated during rapid cell growth. Furthermore, the interaction between cell growth and NO in the PAEC is unidirectional.

Establishment of an immortalized fetal intrapulmonary artery endothelial cell line.

The investigation of fetal pulmonary endothelial cell gene expression and function has been limited by the requirement for primary cells. In an effort to establish an immortalized cell line, ovine fetal pulmonary artery endothelial cells (PAECs; passage 5) were permanently transfected with the E6 and E7 open reading frames of human papillomavirus type 16, and phenotypes related to nitric oxide (NO) production were evaluated up to passage 28. Acetylated low-density lipoprotein uptake, endothelial NO synthase (eNOS) expression, and proliferation rates were unaltered by immortalization. Acetylcholine-stimulated eNOS activity was 218-255% above basal levels in immortalized cells, and this was comparable to the 250% increase seen in primary PAECs (passage 6). eNOS was also acutely activated by estradiol to levels 197-309% above basal, paralleling the stimulation obtained in primary cells. In addition, the expression of estrogen receptor-alpha, which has recently been shown to mediate the acute response in primary PAECs, was conserved. Thus fetal PAECs transfected with E6 and E7 show no signs of senescence with passage, and mechanisms of NO production, including those mediated by estradiol, are conserved. Immortalized PAECs will provide an excellent model for further studies of eNOS gene expression and function in fetal pulmonary endothelium.

Estrogen Acutely Activates Prostacyclin Synthesis in Ovine Fetal Pulmonary Artery Endothelial Cells

Estrogen Acutely Activates Prostacyclin Synthesis in Ovine Fetal Pulmonary Artery Endothelial Cells

Glucocorticoids downregulate cyclooxygenase-1 gene expression and prostacyclin synthesis in fetal pulmonary artery endothelium.

Prostacyclin (prostaglandin I2 [PGI2]) is a key mediator of pulmonary vascular function during early postnatal life, and its production in the pulmonary vasculature rises markedly during that period because of increasing expression of cyclooxygenase type 1 (COX-1). The postnatal rise in COX-1 may be due to the release of inhibition by glucocorticoids, since plasma glucocorticoid levels fall after birth and glucocorticoids decrease PGI2 synthesis in certain nonpulmonary cell types. We therefore studied the direct effects of dexamethasone (DEX) on COX-1 expression in early-passage ovine fetal pulmonary-artery endothelial cells (PAECs). DEX (10(-10) to 10(-6) mol/L) caused a dose-related decrease in COX-1 mRNA expression that was evident by 24 hours, was maximal at 10(-6) mol/L (50% inhibition), and was not due to changes in mRNA stability. There was a parallel decline in COX-1 protein expression. COX-1 protein rose following DEX withdrawal, and DEX blunted the stimulatory effect of 17beta-estradiol on COX-1 expression. DEX alone (10(-8) mol/L for 48 hours) caused a 93% fall in basal PGI2 production, and bradykinin- and A23187-stimulated PGI2 were diminished 96% and 94%, respectively. Similarly, PGI2 synthesis from arachidonic acid fell 86% with DEX; all of the above effects are consistent with COX-1 downregulation. The glucocorticoid receptor (GR) antagonist mifepristone (RU-486; 10(-6) mol/L) blocked the inhibitory effect of DEX, and GR expression was evident by immunoblot analysis. These findings indicate that glucocorticoids downregulate COX-1 expression and PGI2 synthesis in fetal PAECs through the activation of PAEC GR and effects on COX-1 gene transcription. This mechanism may modulate pulmonary PGI2 production in the perinatal period, and it may also play a role in the effects of glucocorticoids on the systemic circulation at a variety of ages.

Estrogen upregulates cyclooxygenase-1 gene expression in ovine fetal pulmonary artery endothelium.

Prostacyclin (PGI2) is a key mediator of pulmonary vasodilation in the perinatal period and its synthesis in the pulmonary vasculature increases markedly during late gestation due to enhanced expression of the rate-limiting enzyme cyclooxygenase-1 (COX-1). The hormone estrogen may play a role in COX-1 upregulation since fetal estrogen levels rise dramatically during late gestation and estrogen enhances PGI2 synthesis in nonpulmonary vascular cells. We therefore studied the direct effects of estrogen on COX-1 expression in ovine fetal pulmonary artery endothelial cells (PAEC). Exposure to estradiol-17beta (E2beta, 10(-)10 to 10(-)6 M) caused a dose-related increase in COX-1 mRNA expression that was evident after 48 h and maximal at 10(-)8 M (fourfold increase). COX-1 mRNA stability was unchanged, suggesting that the upregulation is mediated at the level of transcription. E2beta treatment (10(-)8 M for 48 h) also caused a threefold increase in COX-1 protein expression and a threefold increase in PGI2 synthesis stimulated by bradykinin, the calcium ionophore A23187, or arachidonic acid. The estrogen receptor (ER) antagonist ICI 182,780 fully reversed the effects of the hormone on COX-1 protein expression and on arachidonic acid-stimulated PGI2 synthesis, and ER expression was evident in the PAEC by immunoblot analysis. These findings indicate that physiologic levels of estrogen cause upregulation of COX-1 expression and PGI2 synthesis in fetal PAEC via activation of PAEC ER. This process may play a critical role in optimizing the capacity for PGI2-mediated pulmonary vasodilation at birth, and it may also be involved in estrogen responsiveness in other vascular beds.

Nitric Oxide Upregulates Endothelial Nitric Oxide Synthase Expression in Fetal Pulmonary Artery Endothelial Cells • 1783

Pulmonary vasodilation during cardiopulmonary transition at birth is mediated by endothelium derived nitric oxide (NO), which is generated by the endothelial isoform of NO synthase (NOS), or eNOS. Inhaled NO therapy is currently often used to treat neonatal pulmonary hypertension. Some patients receiving inhaled NO are difficult to wean from the gas after prolonged treatment, suggesting that long-term NO exposure modifies the function of the NO-cGMP signalling cascade. Although NO is known to acutely and reversibly inhibit eNOS activity, the effects of prolonged NO exposure on eNOS expression are not known. We therefore determined the effects of long-term NO exposure on eNOS expression in early passage ovine fetal pulmonary artery endothelial cells (PAEC). To first examine the effects of endogenous NO, PAEC were treated with 2 mM nitro-L-arginine methyl ester (L-NAME). L-NAME caused a decrease in eNOS protein expression that was evident within 8h and maximal by 16h (67% decrease); the effect persisted for at least 48h. The effects of exogenous NO were examined in PAEC treated with the NO donor spermine NONOate or the control parent compound spermine (10-8M to 10-6M). Exogenous NO caused increases in both eNOS protein and cell lysate NOS activity that were also evident within 8h, maximal at 16h (223% of control), and persistent for at least 48h. However, eNOS mRNA abundance was not altered by changes in the level of endogenous or exogenous NO, indicating that the effect of NO on eNOS expression is mediated at the level of translation. This effect is more rapid than previously demonstrated transcriptional regulation of PAEC eNOS by oxygen(≥ 24h). Thus, both endogenous and exogenous NO cause positive feedback, translational upregulation of eNOS gene expression in fetal PAEC. To our knowledge, this is the first demonstration of eNOS regulation mediated at the level of translation. Furthermore, these findings suggest that difficulties with prolonged requirements for inhaled NO in certain patients are not due to changes in the capacity for endogenous NO production.

Shear Stress Regulation Of The Endothelial NOS Gene In Fetal Pulmonary Arterial Endothelial Cells: A Role for Protein Kinase C. • 1936

At birth there is a rapid increase in pulmonary blood flow related to a marked decrease in pulmonary vascular resistance. This causes an increase in shear stress and a resulting increase in both the activity and expression of endothelial NOS (eNOS) in the perinatal lung. The resulting NO released mediates, in part, both the immediate and more gradual decreases in pulmonary vascular resistance seen after birth. However, the regulation of eNOS by shear stress is still poorly understood. The objectives of this study were analyze the regulation of eNOS by shear stress. We exposed cultures of ovine fetal pulmonary arterial endothelial cells (OFPAECs) to increased shear stress using a cone-plate viscometer. This was shown to increase the expression of eNOS mRNA in a time dependent manner with a 4.9-fold increase after 8h. NO production was found to be biphasic, with an initial rapid increase in NO release (0-1h), a plateau phase (1-6h), and then another increase in NO release (6-8h). This second increase in NO release corresponded to the production of new eNOS protein. Since previous studies have implicated protein kinase C (PKC) in shear stressinduced gene expression we determined if PKC played a similar role in eNOS gene regulation. Western blot analysis identified 6 PKC isoforms in OFPAECs: gamma, delta, epsilon, iota, mu, and zeta. Cellular PKC activity increased 7.9-fold in response to 4h of shear stress. PKC inhibition, with four different inhibitors, had little effect on the initial rapid production of NO (0-1h) but all abolished both the induction of endothelial NOS gene expression and the second increase in NO production(6-8h). These results suggest for the first time that shear stress induces eNOS gene expression in a PKC dependent pathway while activationof eNOS protein involves another signal transduction pathway.

Ventilation and oxygenation induce endothelial nitric oxide synthase gene expression in the lungs of fetal lambs.

At birth, ventilation and oxygenation immediately decrease pulmonary vascular resistance (PVR) and increase pulmonary blood flow (PBF); more gradual changes occur over the next several hours. Nitric oxide, produced by endothelial nitric oxide synthase (eNOS), mediates these gradual changes. To determine how ventilation and oxygenation affect eNOS gene expression, 12 fetal lambs were ventilated for 8 h without changing fetal descending aortic blood gases or pH (rhythmic distension) or with 100% oxygen (O2 ventilation). Vascular pressures and PBF were measured. Total RNA, protein, and tissue sections were prepared from lung tissue for RNase protection assays, Western blotting, and in situ hybridization. O2 ventilation increased PBF and decreased PVR more than rhythmic distension (P < 0.05). Rhythmic distension increased eNOS mRNA expression; O2 ventilation increased eNOS mRNA expression more and increased eNOS protein expression (P < 0.05). To define the mechanisms responsible for these changes, ovine fetal pulmonary arterial endothelial cells were exposed to 1, 21, or 95% O2 or to shear stress. 95% O2 increased eNOS mRNA and protein expression (P < 0.05). Shear stress increased eNOS mRNA and protein expression (P < 0.05). Increased oxygenation but more importantly increased PBF with increased shear stress induce eNOS gene expression and contribute to pulmonary vasodilation after birth.

Estrogen upregulates endothelial nitric oxide synthase gene expression in fetal pulmonary artery endothelium.

NO, produced by endothelial NO synthase (eNOS), is a key mediator of pulmonary vasodilation during cardiopulmonary transition at birth. The capacity for NO production is maximal at term because pulmonary eNOS expression increases during late gestation. Since fetal estrogen levels rise markedly during late gestation and there is indirect evidence that the hormone enhances nonpulmonary NO production in adults, estrogen may upregulate eNOS in fetal pulmonary artery endothelium. Therefore, we studied the direct effects of estrogen on eNOS expression in ovine fetal pulmonary artery endothelial cells (PAECs). Estradiol-17beta caused a 2.5-fold increase in NOS enzymatic activity in PAEC lysates. This effect was evident after 48 hours, and it occurred in response to physiological concentrations of the hormone (10(-10) to 10(-6) mol/L). The increase in NOS activity was related to an upregulation in eNOS protein expression, and eNOS mRNA abundance was also enhanced. Estrogen receptor antagonism with ICI 182,780 completely inhibited estrogen-mediated eNOS upregulation, indicating that estrogen receptor activation is necessary for this response. In addition, immunocytochemistry revealed that fetal PAECs express estrogen receptor protein. Furthermore, transient transfection assays with a specific estrogen-responsive reporter system have demonstrated that the endothelial estrogen receptor is capable of estrogen-induced transcriptional transactivation. Thus, estrogen upregulates eNOS gene expression in fetal PAECs through the activation of PAEC estrogen receptors. This mechanism may be responsible for pulmonary eNOS upregulation during late gestation, thereby optimizing the capacity for NO-mediated pulmonary vasodilation at birth.

Estrogen acutely stimulates nitric oxide synthase activity in fetal pulmonary artery endothelium.

Estrogen (E) has nitric oxide (NO)-mediated effects in certain vascular beds, and fetal E levels rise acutely with parturition, suggesting that E may be involved in NO-mediated pulmonary vasodilation at birth. We tested the hypothesis that E acutely stimulates NO synthase (NOS) activity in ovine fetal pulmonary artery endothelial cells (PAEC) by measuring L-[3H]arginine conversion to L-[3H]citrulline in intact cells. NOS activity in the presence of 17 beta-estradiol (E2 beta) rose in a dose-dependent manner, increasing 70-100%, with a threshold concentration of 10(-10) M. This effect was detectable within 5 min of E2 beta exposure, and the maximal response was comparable to that obtained with acetylcholine, which had a threshold concentration of 10(-8) M. Ca2+ removal completely inhibited E2 beta-stimulated NOS activity, and activity with E2 beta and the Ca2+ ionophore A-23187 was not additive. In addition, the expression of the endothelial isoform of NOS (eNOS) was not altered, and the inducible and neuronal NOS isoforms were not detected by immunoblot analysis. These findings indicate that E2 beta acutely stimulates eNOS by Ca2+ influx. Furthermore, E2 beta-stimulated NOS activity was fully inhibited by the E receptor (ER) antagonists tamoxifen and ICI-182,780, and ER mRNA expression was evident in reverse transcription-polymerase chain reaction assays. Thus E acutely stimulates eNOS activity in fetal PAEC via the activation of endothelial ER and increases in intracellular Ca2+.

Glucocorticoids Downregulate Cyclooxygenase-1 Gene Expression and Prostacyclin Synthesis in Fetal Pulmonary Artery Endothelium via Activation of Endothelial Glucocorticoid Receptors ♦ 116

Prostacyclin is a key mediator of pulmonary vasomotor tone and lung function in the early postnatal period. We have previously demonstrated that its production in the ovine pulmonary vasculature rises markedly during early newborn life as a result of increasing expression of the constitutive isoform of cyclooxygenase (COX-1), the rate-limiting enzyme in prostacyclin synthesis. The postnatal rise in COX-1 gene expression may be due to the release of inhibition by glucocorticoids, since plasma glucocorticoid levels fall dramatically after birth and glucocorticoids decrease prostacyclin synthesis in certain nonpulmonary cell types. We therefore studied the effects of dexamethasone (DEX) on COX-1 gene expression in fetal pulmonary artery endothelial cells (PAEC). Early passage ovine fetal PAEC were treated with 10-10 to 10-6M DEX for 24-48 h, and COX-1 mRNA expression was assessed using a semiquantitative reverse transcription-polymerase chain reaction assay. DEX caused a dose-related decrease in COX-1 mRNA expression that was evident by 24 h and maximal at 10-6M (50% inhibition). To determine how this affects PAEC function, prostacyclin synthesis was measured over 60 min in control and DEX-treated cells (10-8M for 48 h). In control cells, basal prostacyclin synthesis was 349±12 pg/well, and synthesis was readily stimulated by bradykinin (7-fold), by the calcium ionophore A23187 (9-fold), and by arachidonic acid (24-fold), each at 10-5M. DEX caused a 93% fall in basal prostacyclin production, and bradykinin- and A23187-stimulated prostacyclin were diminished 96% and 94%, respectively. Furthermore, prostacyclin synthesis from arachidonic acid fell 86% with DEX, all consistent with the downregulation in COX-1 expression. The glucocorticoid receptor antagonist RU486 (10-6M) completely blocked the inhibitory effect of DEX. Thus, glucocorticoids downregulate COX-1 gene expression and prostacyclin synthesis in fetal PAEC through the activation of PAEC glucocorticoid receptors. Loss of this inhibitory effect may contribute to the postnatal rise in COX-1 gene expression in the pulmonary circulation, thereby optimizing the capacity for prostacyclin production in the newborn lung.

Estrogen Acutely Stimulates Nitric Oxide Synthase in Fetal Pulmonary Artery Endothelium via Activation of Endothelial Estrogen Receptors and Calcium-dependent Potassium Channels • 1531

Estrogen (E) has nitric oxide (NO)-mediated vasodilatory effects in specific vascular beds including the pulmonary circulation. Since fetal E levels rise markedly with parturition, E may play a role in the regulation of NO-mediated pulmonary vasodilation at birth. We therefore tested the hypothesis that E stimulates endothelial NO synthase (eNOS) in fetal pulmonary artery endothelial cells (PAEC). To examine the direct effects of the hormone, NOS activity was assessed in cultured ovine fetal PAEC by measuring3 H-L-arginine conversion to 3H-L-citrulline in intact cells over 15 min. NOS activity in the presence of 10-14 to 10 -6M estradiol-17β (E2β) rose in a dose-dependent manner, with a threshold concentration of 10-10M and a maximal increase of 294% at 10-8M E2β. This effect was also detectable within 5 min of E2β exposure (248% increase), and it was comparable to that obtained with acetylcholine (250% increase) which had a threshold concentration of 10-8M. NOS activity in cell lysates was similar in control and E2β-treated cells, indicating that there is not upregulation of NOS expression. The estrogen receptor (ER) antagonist ICI 182,780 fully inhibited E2β-stimulated NOS activity in the intact cells. In addition, ER mRNA expression was evident in PAEC in reverse transcription-PCR assays, and ER protein expression was demonstrated by immunocytochemistry. E2β-stimulated NOS activity was also fully inhibited by removal of extracellular calcium (Ca++), and by LaCl3 (10-4M) and NiCl2 (10-3M), which are inhibitors of Ca++ influx. In contrast, intracellular Ca++ store depletion with thapsigargin (10-6M) had no effect on E2β-stimulated NOS activity. Furthermore, inactivation of Ca++-dependent potassium channels with tetraethylammonium ions (5× 10-4M) fully inhibited E2β-stimulated NOS activity. Thus, physiologic concentrations of E acutely stimulate eNOS activity in fetal PAEC by a mechanism that involves the activation of endothelial ER and Ca++-dependent potassium channels, leading to Ca++ influx. This mechanism may play a role in NO-mediated pulmonary vasodilation at birth, and it may underly E responsiveness in other vascular beds.

Cell Growth Modulates Nitric Oxide Synthase Expression in Fetal Pulmonary Artery Endothelium • 1833

Nitric oxide (NO), produced by the endothelial isoform of nitric oxide synthase (eNOS), is a critical mediator of pulmonary vascular structure and function in the developing lung, causing vasodilation and the inhibition of vascular smooth muscle growth. We have previously shown that pulmonary eNOS gene expression is developmentally regulated during late fetal and early postnatal life, suggesting the eNOS gene expression may be modulated by the state of pulmonary artery endothelial cell (PAEC) growth. We therefore studied the specific effects of cell growth on eNOS expression in cultured ovine fetal PAEC. To examine the effects of cell growth modified by two independent approaches, experiments were performed in cells at varying confluence, and in control cells versus cells stimulated with 20% ovine serum for 24-48 h. Cell proliferation was assessed by examining the incorporation of the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU) into replicating DNA during thymidilate synthetase inhibition with 5-flouro-2′-deoxyuridine. BrdU-positive nuclei were identified by immunocytochemistry with a specific monoclonal antibody. eNOS expression was evaluated by determinations of cell lysate NOS enzymatic activity, which is a highly sensitive measure of changes in enzyme abundance. Compared to 100% confluent cells, cell proliferation was greater at 50% confluence (3.5±0.6% vs. 9.4±0.8% BrdU-positive nuclei, respectively). Greater cell proliferation was associated with attenuated NOS activity, which was 11.9±2.4 vs. 2.3±0.1 pmol/mg prot/min at 100% vs. 50% confluence, respectively. With serum stimulation, cell proliferation increased from 2.8±0.3% to 5.9±0.4% BrdU-positive nuclei. Paralleling the findings with varying confluence, NOS activity was attenuated in the more rapidly-proliferating cells, falling from 2.0±0.3 to 0.5±0.1 pmol/mg prot/min with serum stimulation. These findings contrast with those in systemic endothelium, which exhibit enhanced eNOS expression with greater cell growth. Thus, eNOS expression is markedly attenuated with the activation of cell growth in fetal PAEC. We postulate that eNOS downregulation in rapidly-growing PAEC may facilitate concomitant smooth muscle replication, thereby coordinating the growth of the two cell types during vascularization of the developing lung.

VENTILATION AND OXYGENATION INDUCE ENDOTHELIAL NITRIC OXIDE SYNTHASE GENE EXPRESSION IN THE LUNGS OF FETAL LAMBS. • 1943

With ventilation and oxygenation at birth, a dramatic decrease in pulmonary vascular resistance (PVR) and increase in pulmonary blood flow (PBF) occurs in the first minutes of life which continues more gradually over the next hours. Nitric oxide (NO) mediates these gradual changes because they are blocked by the administration of NO synthesis inhibitors. In the vasculature, NO is produced from L-arginine by endothelial nitric oxide synthase (eNOS). To determine how ventilation, oxygenation, and the resulting increase in PBF affect eNOS gene and protein expression, near-term fetal lambs (138-140 days' gestation) were ventilated in utero with a hypoxic gas (without changing fetal arterial blood gases) or with 100% O2. Vascular pressures and left PBF were measured. After 8 hrs of ventilation, the lungs were harvested. Total RNA and protein were prepared. RNase protection assays, Western blotting, and in situ hybridization were performed. Lungs from unventilated fetal lambs were controls. Hypoxic gas ventilation (N=3) increased PBF from 3.8±3.4 to 9.4±9.0 ml/min/kg and decreased PVR from 33.8±34.7 to 18.5±22.7 mm Hg/ml/min/kg. 100% O2 ventilation (N=4) increased PBF from 6.8±7.9 to 78.3±29.4 ml/min/kg and decreased PVR from 32.2±43.6 to 0.7±0.3 mm Hg/ml/min/kg. Hypoxic gas ventilation only slightly increased eNOS expression; 100% O2 ventilation increased eNOS gene expression 3-fold without changing protein levels. To define whether these changes in gene expression were due to changes in oxygenation or to the increase in pulmonary blood flow and the resulting increase in shear stress, fetal pulmonary artery endothelial cells were exposed to 1, 21 and 95% oxygen (for 8-24 hrs) or to 25 dynes/cm2 of shear stress (for 8 hrs). 95% oxygen increased eNOS gene expression 3-fold, while shear stress increased it by 20-fold. Increased oxygenation and increased pulmonary blood flow with increased shear stress rapidly induce eNOS gene expression and contribute to pulmonary vasodilation after birth.

Oxygen upregulates nitric oxide synthase gene expression in ovine fetal pulmonary artery endothelial cells.

Nitric oxide (NO) is critically involved in oxygen-mediated pulmonary vasodilatation in the fetus and newborn. We determined the effects of prolonged alterations in oxygenation on endothelial NO synthase (eNOS) gene expression in early passage ovine fetal intrapulmonary artery endothelial cells (PAEC). PAEC were exposed to PO2 = 50 or 150 mmHg for 48 h, and eNOS protein expression was evaluated by immunoblot analysis. eNOS protein expression was 2.7-fold greater at higher oxygen tension; eNOS upregulation was also evident after 24 h. Inducible NOS protein was not detectable by immunoblot at either level of oxygenation. In the lung, the effect of oxygen on eNOS expression may be specific to the endothelium, as eNOS expression in bronchiolar epithelial cells of Clara cell lineage was not altered by varying oxygen tension. The oxygen-related increase in eNOS protein in the fetal PAEC was associated with 2.5-fold greater NOS enzymatic activity. In parallel, there was a 2.8-fold rise in eNOS mRNA abundance. Thus eNOS gene expression in ovine fetal PAEC is upregulated by oxygen, and this is mediated at the level of gene transcription or mRNA stability. This process may play an important role in oxygen modulation of pulmonary vasomotor tone in the fetus and newborn.